The present invention relates to improvements in analytical techniques. The invention has particular utility in connection with determination of low levels of analytes in highly complex mixtures, and will be described in connection with such utility, although other utilities are contemplated.
The determination of very low levels of specific analytes in complex mixtures of hundreds of possible interferences is a classical problem in analytical chemistry. For example, in recent developments in the assessment of oxidative stress and in the assessment of genetic damage from inherited and environmental factors, the measurement of urinary 8 hydroxy 2xe2x80x2 deoxyguanosine (8OH2xe2x80x2dG) and other similar purine and pyrimidine indicators of DNA damage has developed as a potentially useful diagnostic therapy directing tool. These measurements have been used in clinical research in the areas of neurological disorders, cardiovascular problems, cancer, and assessment of biological environmental risk.
The overall utility of such measurements is compromised, however, by the lack of reliable measuring technology that will allow the intercomparison of values among different laboratories and studies. While several methods incorporating multiple separations and finally analytical techniques of liquid chromatography with electrochemical detection or gas chromatography, or mass spectroscopy have been published, they all suffer from problems of reliability, certainty of the analyte measured, complexity of preparation and manipulation, short and long term accuracy and precision, and cost. One of the most commonly cited techniques has been liquid chromatography with electrochemical detection employing a variety of preparation and concentration procedures and/or automated column switching. In automated column switching a portion of the eluent band from a first column is trapped in an injection loop and then transferred to a second column with different characteristics of separation. While some researchers reportedly have obtained reliable information with these techniques, the procedures are both fragile and prone to individual specific errors. This is because of the small non-quantitative and variable amounts trapped from the band and from the first column (typically less than 10%), and because of the highly variable nature and high level of interfering species at the typical levels of ca 5 ng/ml of analyte of interest.
It is thus an object of the invention to overcome the aforesaid and other disadvantages of the prior art. Another object of the present invention is to provide an analytical technique for determining low levels of analytes in complex mixtures. A more specific object of the invention is to provide an analytic technique for analyzing 8 hydroxy 2xe2x80x2 deoxyguanosine in biological samples.
In order to effect the foregoing and other objects of the invention, in accordance with the present invention, there is provided a sample pre-separation and concentration system comprising a plurality of trapping columns having a high selectivity for classes of compounds of interest, upstream of a standard separation column. For example, as applied to the analysis for 8OH2dxe2x80x2G in urine and other biological samples, the basis of the invention is the replacement of the injection loop from the first column with one or more small porous carbon columns that have a very highly selectivity for purines, and certain other classes of compounds such as aromatic amines and nitro compounds and certain flavones flavenoids and other highly conjugated species. These columns have sufficient selectivity that they can trap essentially the entire eluting band containing the analyte of interest in a very small, e.g. 20-250 ul volumes, and then be flushed for a relatively long time by a second eluting buffer to remove almost all species but the analyte of interest. The porous carbon column is then switched to the head of a second separation column employing a buffer identical to the flushing buffer except for a displacing agent that strips the analyte of interest from the carbon in a sharp peak that is compatible with optimum separation characteristics of the second column. By way of example, for 8OH2xe2x80x2dG, the displacing agent of preference is Adenosine, a compound of similar structure but not electroactive. The principle of similarity by non-detectability is general for the third buffer additive. For example, phenylalanine may be used to displace nitrotyrosine.